The purpose of this project is to define the cell lineages of a model marsupial, the gray short-tailed opossum (Monodelphis domestica) during cleavage, blastocyst formation, and gastrulation. A fundamental understanding of developmental mechanisms in mammalian embryogenesis will have significant impact on human embryonic wastage and birth defects. However, this goal cannot be achieved by analysis of placental mammals in isolation form mammalian evolution. Because of their close relatedness to placental mammals and unique morphological aspects of their early embryogenesis, marsupial embryos offer an unprecedented opportunity for understanding important issues of mammalian embryogenesis, including the basis for differentiation of embryonic and extraembryonic tissues, the fate map for allocation of cells to the primary germ layers and organ rudiments during gastrulation and the relevance of DNA methylation as a molecular mechanism for genomic imprinting. Initial descriptive studies will establish a normal developmental table for this species and refine culture conditions of cleavage and gastrula sages. Cell lineage analysis will be performed using microinjected tracers at cleavage and gastrula stages. Cell lineage analysis will be performed using microinjected tracers at cleavage and gastrula stages to determine the fate of individual blastomeres in forming the unilaminar marsupial blastocyst, and of individual epiblast cells in forming the primary germ layers and organ rudiments. The gastrula study should provide a more complete clonal fate map than of any other vertebrate species owing to the easy accessibility of the epiblast layer on the outer surface of the embryo. Analysis of the extent of DNA methylation of repetitive and unique sequence DNA of embryonic and extraembryonic tissues as well as adult soma and germ line using restriction enzyme digestion and southern blotting will be informative about the role of methylation in genomic imprinting. Moreover, analysis of the extent of imprinting in marsupials by experimental induction of parthenogenesis should reveal the evolutionary origins and significance of imprinting. This project should thus reveal basic principles of mammalian development relevant to human health by taking advantage of the prospective provided by marsupials.